Molecular diagnostics has been a routine clinical laboratory procedure for biological detection and monitoring. A specimen of a biological sample coming from various biological sources usually contains a mixture of chemicals, metabolites, macromolecules, cells, virions, organisms, and nucleic acid sequences. Typically, two common challenges exist for detection and monitoring: background interference and limits of detection (LOD). Namely, the target biological samples are usually present in a very small amount compared with other background components in the biological sample. Such non-target background components may interfere with the detection downstream. LOD becomes an issue when not enough copies of the target exist in the sample. The same LOD challenge is the same as in chemical analysis, which usually requires highly sophisticated instruments for conducting analysis work. Examples include using mass spectrometry for detection of bis(n-butyl)phthalate plasticizer contamination in the food supply chain, melamine contamination in milk, cadmium and heavy metal contamination in the soil or food. The background interference is an order of magnitude higher than the contaminants. On-site analysis is usually difficult without extensive treatment in the laboratory involving instruments such as a GC spectrometer or mass spectrometer, for example.
Background interference is usually solved by purifying the sample during the preparation stage. Targets are captured and background components are removed by one or more washing procedure(s). Typical examples include: Enzyme-linked Immunosorbent Assay (ELISA) and different chromatographic methods. Lateral flow platforms can also be used to capture targets while background components are removed from the immobilized targets. Another example of a purification procedure for nucleic acid targets are purification kits such as the Qiagen QIAamp DSP DNA Blood Mini Kit.
The LOD issue for nucleic acids is usually resolved by amplification of the sample target first, which allows later detection by fluorescence emission, electrical and electronic methods, e.g. voltametry, current measurement, capacitance measurement, or impedance spectroscopy. Among these detection or amplification methods, electrical power is required to provide the light or conduct the electrical/electronic detection. One example of resolving the LOD issue with low abundance nucleic acid targets is by amplifying nucleic acid targets in vitro by using the polymerase-chain reaction (PCR). The signal associated with the presence of the target sequence can be further amplified by fluorescence methods. Each PCR amplicon that is labeled by fluorescent tags(s) can produce 1000 or 10,000 more protons in a single excitation/measurement period when a fluorescence beacon or probe is used to detect the amplicon. Another example is a Campylobacter-like organism test, in which H. pylori cells multiplies in the culture and is thus amplified, and where urease secreted from the amplified cells can be detected after the number of cells passes the detection threshold. Yet another example is the screening of MRSA. This detection method differentiates the colonies of the multiplied and amplified cells on a culture medium.
However, current methods which rely on removal of various combinations of background interference and amplification detection have not filled the need for a robust, cost effective, rapid, easy to use detection method for targets, which is compatible in resource limited environments. The requirement for an electrical power source to perform the current tests by powering the instrument or providing the temperature incubation has restricted access to this technology in resource-poor regions or conditions. The time required to practice the current methods is long. Many hours or days are required to produce enough biological targets to generate sufficient detection signals. The amplification step used for pathogen detection, metabolite determination, or nucleic acid sequence detection usually requires expensive laboratory instruments and trained specialists to run the instruments. In the case of PCR, care is required to handle labile reagents, and special diligence is essential to avoid contamination between samples. Moreover, the instruments required to perform PCR are expensive and complex. The above considerations are severe limitations that prevent POC (point of care) use or provide rapid sample-to-result in less than 30 minutes. The present invention provides solutions to these and other needs.